Feature architecture aware phylogenetic profiling indicates a functional diversification of type IVa pili in the nosocomial pathogen Acinetobacter baumannii

The Gram-negative bacterial pathogen Acinetobacter baumannii is a major cause of hospital-acquired opportunistic infections. The increasing spread of pan-drug resistant strains makes A. baumannii top-ranking among the ESKAPE pathogens for which novel routes of treatment are urgently needed. Comparative genomics approaches have successfully identified genetic changes coinciding with the emergence of pathogenicity in Acinetobacter. Genes that are prevalent both in pathogenic and a-pathogenic Acinetobacter species were not considered ignoring that virulence factors may emerge by the modification of evolutionarily old and widespread proteins. Here, we increased the resolution of comparative genomics analyses to also include lineage-specific changes in protein feature architectures. Using type IVa pili (T4aP) as an example, we show that three pilus components, among them the pilus tip adhesin ComC, vary in their Pfam domain annotation within the genus Acinetobacter. In most pathogenic Acinetobacter isolates, ComC displays a von Willebrand Factor type A domain harboring a finger-like protrusion, and we provide experimental evidence that this finger conveys virulence-related functions in A. baumannii. All three genes are part of an evolutionary cassette, which has been replaced at least twice during A. baumannii diversification. The resulting strain-specific differences in T4aP layout suggests differences in the way how individual strains interact with their host. Our study underpins the hypothesis that A. baumannii uses T4aP for host infection as it was shown previously for other pathogens. It also indicates that many more functional complexes may exist whose precise functions have been adjusted by modifying individual components on the domain level.

Introduction Acinetobacter baumannii is a Gram negative nosocomial pathogen. A recent world-wide survey estimated that antimicrobial resistant A. baumannii strains were responsible for 10.4% of bacterial infections with fatal outcome in 2019 [1]. The spread of multi-or even pan-resistant A. baumannii isolates [2][3][4] is accompanied by a surge in virulence [4][5][6][7][8], and thus novel therapeutic treatments are necessary for sustainable infection management. Over the past years, experimental studies integrated with comparative genomics analyses have sought to identify genetic determinants of A. baumannii virulence [9][10][11][12][13][14][15][16][17][18][19][20][21]. The resulting candidates are involved in a broad spectrum of biological processes including NOS and ROS resistance and metabolic adaptation, but some also indicate changes in the way the pathogen interacts with its environment [15,21].
Pili, or 'hair-like' surface appendages, are main mediators of bacterium-environment interaction [22]. Most bacterial phyla possess type IV pili (T4P) [23,24], multi-purpose nanomachines that act via dynamic cycles of extension and retraction mediated by cytoplasmic motor ATPase-driven polymerization and depolymerization of pilin subunits [25][26][27][28][29][30]. To date, three sub-types of T4P are known of which sub-type 'a' is most prevalent [24]. T4aP are involved in a variety of functions [22]. Among these, surface adhesion, bacterial motility and the uptake of environmental DNA are tightly connected to bacterial virulence [31,32]. It is thus not surprising that several Gram negative and Gram positive human pathogens use T4aP for processes connected to host infection [31,[33][34][35][36][37][38]. In Acinetobacter, T4aP play a role in cell adhesion [39], twitching motility and natural transformation [28,[40][41][42]. Moreover, the two-component regulatory system BfmRS, which is important for survival of A. baumannii in a murine pneumonia model, also controls T4aP production [43]. While this suggests that T4aP could also drive A. baumannii virulence, the most comprehensive comparative genomics study so far between pathogenic and a-pathogenic Acinetobacter species failed to detect differences that could hint towards lineage-specific changes in T4aP formation or function [21].
T4aP are prevalent in bacteria irrespective of their lifestyle [22]. Their recurrent recruitment by pathogens for processes connected to host infection therefore suggests that only considerably subtle modifications are necessary to transform T4aP into a virulence factor. Therefore, any adaptive changes in pathogenic Acinetobacter might have escaped the attention thus far, because they require higher resolving analyses beyond determining the presence/ absence of T4aP components. Indeed, structural variants of the major pilin subunit PilA were recently detected in A. baumannii strains. This suggested the existence of functionally diverse T4aP in this species [39,44]. A comprehensive analysis on this level of resolution for all T4aP components considering, at the same time, the wealth of sequence data covering the full range of Acinetobacter diversity is missing. Therefore, it is still unclear to what extent T4aP differ between members of this genus and what consequences this may have for the interaction of A. baumannii with the human host.

PLOS GENETICS
Here, we increase the resolution of the comparative analysis of T4aP components to the level of individual protein features, such as the presence of Pfam-or SMART domains, of transmembrane domains and of low complexity regions [45]. For each protein, we integrate the annotated features from N-to C-terminus into a feature architecture and compare these between orthologs of the T4aP components. We then exploit that differences in the feature architectures of two orthologs serve as a proxy of their functional divergence [45]. To thoroughly chart the extent of variation in the precise design of 20 T4aP across the genus Acinetobacter, we integrated their genus-wide phylogenetic profiles across more than 884 bacterial isolates with an assessment of feature architecture similarity. Three candidates organized in the same gene cluster have altered feature architectures in most pathogenic Acinetobacter isolates, which indicates a change in function. A subsequent evolutionary characterization integrated with modelling of their 3D structures and a downstream experimental characterization identifies the pilus tip adhesin ComC as the most prominent candidate driving the functional diversification of T4aP in pathogenic Acinetobacter. In summary, our results provide first-time evidence that pathogenic Acinetobacter have modulated the precise function of T4aP by changing the structural layout of the pilus tip adhesin.

Phylogenetic profiles of Type IV pilus components
Acinetobacter T4aP components are best characterized in the naturally transformable bacterium A. baylyi ADP1 (Fig 1A), and we used the protein set provided by Averhoff et al. 2021 [46] to prime our analysis. We additionally considered the prepilin peptidase PilD because the corresponding gene is part of the pilBCD operon in A. baylyi, and because it is likely involved in T4aP biogenesis [40,41]. All A. baylyi ADP1 T4aP components are represented in the A. baumannii type strain Ab ATCC 19606 T (Table 1), which allowed to reconstruct the evolutionary history of T4aP from the perspective of A. baumannii. We first performed a targeted ortholog search for the individual Ab ATCC 19606 T T4aP components across 855 Acinetobacter isolates that cover the diversity of the genus complemented with 29 outgroup species. The resulting presence/absence patterns of orthologs are summarized in the phylogenetic profiles shown in Fig 1B. Orthologs to all T4aP components are found throughout the genus Acinetobacter. In the outgroup species, however, the phylogenetic profiles become more sparsely filled. To investigate possible reasons for the non-detection of orthologs in these species, we focused on the gene triple comB, pilX and comC. The three genes reside next to each other in the genome of Ab ATCC 19606 T where they are flanked by pilV and comE. In Pseudomonas aeruginosa PAO1, orthologs were identified only for pilV and comE (see Fig 1B), but like the situation in Ab ATCC 19606 T , the two corresponding genes flank several T4aP components [47]. Of these, two show a weak albeit significant amino acid sequence similarity to ComB and ComC from A. baumannii respectively (S1 Fig). For Ab ATCC 19606 T PilX, we found no  Table 1. (B) Phylogenetic profiles of the Ab ATCC 19606 T T4aP components across 884 bacterial isolates representing 83 named species. Taxa are summarized on the species level. A dot indicates the presence of an ortholog in the respective species, and the dot size represents the fraction of the subsumed isolates an ortholog was identified in. The color encodes the median feature architecture similarity (FAS score) between the protein in Ab ATCC 19606 T and its orthologs within a species. The dot color gradient (FAS_F; blue to orange) captures architecture differences using the feature architecture of the Ab ATCC 19606 T protein as reference. The score decreases if features seen in the Ab ATCC 19606 T protein are missing in the respective orthologs. The cell color gradient (FAS_B; white to pink) captures architecture differences using the feature architecture of the ortholog as reference. The score decreases if features seen in the ortholog are missing in the Ab ATCC 19606 T protein. Taxa are ordered according to increasing phylogenetic distance to A. baumannii using the information provided in [21] and [100]. 'γ' and 'β' represent γ-and βproteobacteria, respectively. The full data is available as S1 Data. (C) Pfam domain architectures of the T4aP components in Ab ATCC 19606 T . Protein lengths are not drawn to scale. Pfam accessions and domain descriptions are available in S1 homolog in Pa PAO1 by means of sequence similarity. However, in both species a gene annotated as pilX is placed at an identical position in the gene clusters of the two species (see S1 Fig). Integrating all evidences indicates that all three A. baumannii genes have orthologs in P. aeruginosa, which have been overlooked due to their extensive sequence divergence. Thus, at least part of the gaps in the phylogenetic profiles of T4aP components is due to a limited sensitivity of the ortholog search [48].

Feature architecture changes in T4aP components
The phylogenetic profiles provide no evidence for a lineage-specific modification of T4aP within Acinetobacter that is driven by the gain or loss of individual genes. We therefore increased the resolution of the analysis by comparing the feature architectures of the Ab ATCC 19606 T T4aP components to those of their orthologs (Fig 1B-1D). For most proteins, feature architectures are either conserved across the genus, or differ only in the presence/ absence of low complexity regions or coiled coil regions (see S2 Fig). However, the feature architectures of five proteins, PilX, FimT, PilV, FimU, and ComC deviate to an extent between orthologs that a functional diversification is conceivable [45]. Of these proteins, PilX and FimT are unlikely to drive T4aP diversification on a larger scale. The feature architectures of many PilX orthologs differ from that of the protein in Ab ATCC 19606 T by the absence of a PilX N-term Pfam domain (PF14341; see Fig 1C and 1D). Upon closer inspection we found

Evolutionary histories of ComC, FimU and PilV in Acinetobacter
Differences in the feature architectures of ComC-, FimU-, and PilV orthologs are first indications for a functional diversification of T4aP components within Acinetobacter. We next investigated the evolutionary histories of the three proteins in greater detail. A phylogenetic analysis of the ComC orthologs revealed that the species A. baumannii and the genus Acinetobacter are both paraphyletic regarding this locus (Fig 2 and S7 Fig). To exclude the possibility that the paraphyly is an artefact of insufficient phylogenetic signal in the data, we confirmed that an alternative tree topology with monophyletic Acinetobacter isolates explains the data significantly worse (AU test; p = 0.002; [50]). In the genome of Ab ATCC 19606 T , all three genes (comC, fimU and pilV) reside in close vicinity (see Fig 3). To determine whether they are not only physically but also genetically linked, we labeled each taxon in the ComC tree with the respective variant combination for ComC, FimU and PilV. Within A. baumannii, but also for most isolates from the Acinetobacter calcoaceticus-baumannii (ACB) complex, ComC Var1 is almost exclusively found together with FimU Var1 and PilV Var1 . ComC Var2 is typically associated with FimU Var2 and PilV Var2 (Fig 2 and S7 Fig). The association of the variants is only broken up in early branching Acinetobacter species outside the ACB complex. Interestingly, the tree reveals a third clade comprising individual A. baumannii isolates and one representative of A. calcoaceticus. The feature architectures of the three candidate proteins in this clade resemble that of Var1 (S8 Fig), but a higher resolving analysis reveals differences in the case of ComC (see below). To give credit to the distinct phylogenetic placement of this clade, we refer to it as Var1-2 to distinguish it from the more abundant Var1-1.
The phylogenetic analysis has revealed that ComC, FimU and PilV together exist as three evolutionarily distinct lineages with two alternative feature architecture layouts in A. baumannii. This suggests that recombination may have affected the evolution of this locus. We next used the change in the pattern of shared variants between the bacterial isolates to assess the length of the genomic region that was likely involved in these recombination events [51]. We included 5 kbp upstream and 3.5 kbp downstream of ComC in the analysis such that the investigated genomic region harbors four additional T4aP components as well as six flanking genes with different functions (Fig 3). To rule out that changes in gene order represent a physical barrier to recombination, we confirmed that the order of these 13 genes is conserved across the Acinetobacter diversity (S9 Fig). The analysis revealed that the recombination block spans all seven T4aP components in this region but excludes the flanking genes. This observation integrates well with the finding that the gene tree of the concatenated T4aP components leaves A. baumannii paraphyletic (see Fig 3), whereas the gene tree based on a concatenation of the five flanking genes supports monophyletic A. baumannii (S10 Fig).
Integrating the results of the evolutionary analyses with the feature architecture variant assignments for ComC, FimU and PilV reveals that the entire cluster of T4aP associated genes represents an evolutionary cassette. The different domain architecture layouts of the three proteins characterize two main variants of this cassette, and an exchange of this cassette occurred at least twice during A. baumannii diversification.

3D modelling of ComC reveals variant-specific structural variation
In the highest resolving analysis, we assessed how the differences seen between orthologs of ComC on the feature architecture level are reflected in the predicted 3D structures (Fig 4 and S11 Fig; see S12 and S13 Figs for FimU and PilV, respectively). ComC is characterized by the presence of two globular domains that are connected by a linker (Fig 4A). The C-terminal domain shows considerably little structural variation across the investigated proteins (see Fig  4B panel 3). It comprises the part of the ComC sequence that is consistently annotated with the Neisseria_PilC Pfam (PF05567) domain across all ComC orthologs (see Fig 1C). In contrast, the N-terminal half of ComC is structurally more variable (see Fig 4B panel 2), and this does not coincide with a greater uncertainty in the model accuracy for this part of the predicted 3D structure (see S11 Fig). In ComC Var1-1 , the N-terminal part is predicted to fold into an α/β doubly wound open twisted beta sheet conformation, which is surrounded by 7 parallel alpha helices arranged in a cylindrical conformation and an external alpha helix (S14A Fig). This fold agrees with previous structural characterization of the von Willebrand factor type A (vWFa) domain [52,53], and of the vWFa domain in integrin α II b (PDB: 3NIG). A similar structural layout is predicted also for ComC Var1-2 (S14B Fig), and both findings are consistent with the annotation of a Pfam VWA_2 domain in the N-terminal part of these variants. However, ComC Var2 forms a similar fold (S14 C), and all three variants share the presence of a  share the presence of a finger-like protrusion harboring a Tyr-rich motif. Note that these fingers are embedded into the vWFa domains of both variants, however they are differently positioned both in the predicted structures ( Fig 4A and 4B) and in the respective amino acid sequences (Fig 4C and S14A and S14B Fig). Therefore, the two fingers are very likely of different evolutionary origins although their structural similarity and the shared presence of the Tyr-rich motif suggest that they originated from the same source (Fig 4D and 4E). Neither ComC Var2 in A. baumannii or in A. baylyi, nor PilY1 in P. aeruginosa are in possession of a similar protrusion (Fig 4A and 4B). However, the VAST analysis revealed that these proteins carry other insertions in the region that likely forms a vWFa domain (see S15 Fig).

Functional role of ComC in A. baumannii
The in-silico analysis has provided substantial evidence for a hitherto unknown diversity of T4aP within A. baumannii. A prominent driver of this diversity is the pilus tip adhesin ComC, and here specifically the N-terminal region that folds into a vWFa domain. ComC Var1 differs from ComC Var2 by the presence of a finger-like protrusion that has been integrated into the vWFa domain. Moreover, ComC Var1-1 displays a local structural similarity to mechanosensitive β3-integrins [56] (see S15 Fig), which belong to a superfamily of cell adhesion receptors in animals [57]. With the following experiments, we shed initial light on the functional relevance of the vWFa domain variant in ComC Var1-1 and of the finger-like protrusion therein. We created three different ComC constructs: the full-length version of Ab ATCC 19606 T comC, a truncated version that lacks the subsequence that is annotated with the Pfam VWA_2 domain (comCΔVWA), and a version where we exclusively deleted the region that encodes the finger in ComC Var1-1 (comCΔ166-256). Note that a comparison of the predicted structures for ComC and for the ComCΔ166-256 mutant provided no evidence for a mis-folding of the mutant (S16 Fig). The subsequent experiments were performed in a comC knock-out mutant of Ab AYE-T, because Ab ATCC 19606 T did neither twitch nor to take up environmental DNA in our hands.
We initially confirmed that Ab AYE-T ΔcomC strain showed no noticeable piliation defect ( Fig 5A). This finding is consistent with the observations that a comC deletion has no effect on piliation in A. baylyi [58] and in Neisseria [59], and we conclude that a deletion of comC does not interfere with piliation in any A. baumannii isolate. Subsequently, we investigated the role of ComC and of the vWFa domain in host cell adhesion (Fig 5B). Compared to wild-type Ab AYE-T, a comC knock out mutant (Ab AYE-T ΔcomC) displayed a significantly reduced structural conservation score ranging from 1 (structurally identical) to 0 (no similarity). ID: percent of sequence identity in the structural alignment. (C) Multiple sequence alignment of the N-terminal part of representatives for the three ComC variants in A. baumannii. The alignment covers the amino acids 24-233 of Ab ATCC 19606 T ComC. The sequences forming the finger-like protrusions in ComC Var1-1 and ComC Var1-2 are shaded in brown. The sequences for the three variants represent the corresponding species shown in (A). (D) Structural similarity between the finger-like protrusions of ComC Var1-1 and ComC Var1-2 . The color gradient from blue to red indicates decreasing similarity. The tyrosine-rich motif is highlighted in green. (E) Pair-wise sequence alignment between the sequences forming the finger-like protrusion in ComC Var1-1 and ComCVar 1-2 . Conserved residues are indicated in dark blue; the tyrosine-rich motif is shown in green.
https://doi.org/10.1371/journal.pgen.1010646.g004 adhesion rate to HUVECs (n = 4; t test: p<0.05). Complementing the mutant with the full length comC increased the adhesion rates significantly (n = 4; t test: p<0.05), whereas no significant increase was observed when we used the either comCΔVWA or comCΔ166-256 for complementation. We next investigated the role of ComC in T4aP mediated twitching, and for natural transformation. Ab AYE-T ΔcomC showed no twitching motility, and this phenotype was at least partly restored upon complementation with the full length comC (Fig 5C). Notably, neither of the truncated comC mutants could restore the capability to twitch to a noticeable extent. Like the effect on twitching motility, the deletion of comC abolished natural transformation (Fig 5D). Complementation with the full length comC almost fully restored the phenotype. Interestingly, this time the complementation with comCΔVWA and comCΔ166-256 also restored natural transformation, however with frequencies that are an order of magnitude below those of the full length comC. The partial restoration of natural competence is an independent albeit indirect indication that the truncated ComC mutants do not show a strong piliation defect. To provide further evidence that the observed phenotypes are also not the effect of more subtle pilus instabilities caused by a truncated ComC, we complemented Ab AYE-T ΔcomC with the wild-type ComC Var2 from Ab 17-VT4715T-2. We investigated only the effect of the complementation on natural competence and on twitching, as these phenotypes showed the strongest effect upon comC deletion. Notably, ComC Var2 could neither restore natural competence nor twitching (Fig 5C and 5D).  Table). '*' indicates a significant difference (one tailed t test: p<0.05). Complementation with the truncated comC mutants did not significantly increase adhesion rates. The experimental data provide first-time evidence that the N-terminal half of ComC Var1-1 and the vWFa domain variant contained therein play a critical role in T4aP mediated adhesion to HUVEC cells, for twitching and at least contribute to natural competence. While both twitching and natural competence was abolished upon comC deletion, the ability to adhere to HUVEC cells was only reduced. This is best explained by the effect of other adhesins, such as ATA [60], Csu fimbriae [60], or InvL [61], which all contribute to host cell adherence. Interestingly, we observe for all three tested functions the same phenotypic effect when deleting only the finger-like protrusion that is characteristic for ComC Var1-1 . Thus, that at least part of the ComC Var1-1 function seems conveyed by this finger. In line with this hypothesis, we find that ComC Var2 cannot rescue a ComC Var1-1 knock-out

Discussion
Type IVa pili are prevalent in bacteria irrespective of their lifestyle where they convey a broad range of functions [22,24]. In some and often only distantly related human pathogens, they represent key virulence factors [32,33,62,63]. This indicates that the precise functions of T4aP have changed multiple times during evolution and probably as an adaptation to differing habitats and lifestyles. The genus Acinetobacter harbors environmental bacteria, bacteria that colonize various animals, as well as human pathogens [64]. This diversity in lifestyles provides an optimal setup for tracing also subtle genetic changes that underlie the functional diversification of T4aP that are used by the bacteria to interact with their environment.
The individual components necessary for building up the T4P machinery are almost ubiquitously present across the Acinetobacter diversity. This indicates that missing even one factor most likely renders the entire pilus dysfunctional. Along the same lines, it suggests that T4aP are essential for Acinetobacter fitness independent of both habitat and lifestyle. However, conspicuous differences between individual T4aP components across Acinetobacter isolates emerged on the level of their feature architectures. The connection between feature architecture of a protein and its function is well documented (e.g., [45, [65][66][67][68]. Therefore, the differences for PilV, FimU and, more prominently for the pilus tip adhesin ComC between Acinetobacter isolates point towards a lineage-specific modification of T4aP function. T4a pilus tip adhesins (T4a-PTA) have received considerable attention in various bacterial species, among them several human pathogens [59,[69][70][71]. Thus far, all investigated proteins share the presence of a Ca 2+ binding domain in the C-terminal half (Pfam: Neisseria_PilC; PF05567) and assume similar roles in basal pilus function [59,69,[72][73][74][75]. However, the precise functions of the N-and C-terminal globular domains differ among species. In Neisseria, the N-terminal half of PilC1 mediates host cell adherence [59,69,73,74]. In Legionella, it is necessary for host cell invasion [76], and in P. aeruginosa, it has been associated with the mediation of surface adhesion, mechanosensing, and regulation of pilus retraction [36,71]. In both Legionella and P. aeruginosa, the host cell adhesion function is mediated by the C-terminal half of the protein [76,77].
Here, we have shown that the most prominent differences between the individual ComC orthologs both within A. baumannii and across the genus reside in the N-terminal part. ComC proteins of most A. baumannii isolates together with that of some members of the ACB could be exclusively annotated with the VWA_2 Pfam domain. This indicates the presence of a von Willebrand Factor A domain (vWFa) [78], a domain that mediates cell adhesion and cell migration in eukaryotic proteins in [79]. A vWFa domains in a bacterial PTA was first described for the Pi-2a pilus in the Gram-positive Streptococcus agalactiae, a leading cause of sepsis and meningitis. As hypothesized from its function in eukaryotes, this domain indeed mediates host cell adhesion [80]. Subsequently, a vWFa domain was also found in PilY1 of P.
aeruginosa [36,70,81], and supporting its role in bacterial virulence, it was subsequently found that vWFa domains can activate macrophages, central regulators of airway inflammation [82,83]. On the first sight, our findings that exclusively ComC of pathogenic Acinetobacter could be annotated with a Pfam VWA_2 domain suggest that the acquisition of a vWFa domain drives the conversion of T4aP into a virulence factor.
However, the situation appears more complex. Higher-resolving analyses indicate that also ComC of a-pathogenic Acinetobacter isolates possess a vWFa domain although the corresponding amino acid sequence is not similar above threshold to the VWA_2 Pfam domain. Evidences include the presence of the characteristic metal ion-dependent adhesion (MIDAS) motif (see S14 Fig; [54]) as well as extended stretches of local structural similarity between the N-terminal region of ComC and eukaryotic vWFa containing proteins (see S15 Fig). What however differentiates the investigated ComC variants are independent insertions into the conserved structural scaffold formed by a vWFa domain (see S14 and S15 Figs). In ComC Var1 such an insertion resulted in the formation of a finger-like protrusion (see Fig 4 and S15 Fig). Thus, rather than the presence of a vWFa domain in the PTA it might be the variant of the vWFa domain that determines whether T4aP are involved in virulence related functions, or not.
Testing the function of ComC Var1-1 in-vivo revealed that this protein is involved in host cell adhesion, twitching and DNA uptake, and that these functions are conveyed by the N-terminal half and of the vWFa domain therein. Interestingly, a deletion of the finger-like protrusion in ComC Var1-1 was sufficient for impairing all three processes to an extent that is comparable to the deletion of the entire vWFa domain. Because structural modelling revealed no indication that the deletion of the finger results in misfolding of ComC (see S16 Fig), these findings suggest a functional role of this structure. The observation that wild-type ComC Var2 could not complement the deletion of ComC Var1-1 further supports this view. Still, our evidence is only preliminary, and further analyses will be necessary to prove the involvement of this finger in ComC Var1-1 function. It will then also be interesting to see whether ComC Var1-2 differs in function from ComC Var1-1 , and whether the Tyr-rich motif, that is present in the fingers of both variants has a functional role. Tyrosine assumes a broad spectrum of functions in natural systems, and short tyrosine rich peptides display a high propensity for self-assembly [84]. A functional role of this motif in ComC Var1 is therefore conceivable.
Next to ComC, also FimU and PilV vary both in their feature architecture and in the inferred 3D structure across the investigated isolates. The respective variants are tightly associated with the two main ComC variants forming different layouts of the T4aP cassette. The exchange of this cassette, which happened at least twice during A. baumannii diversification, likely resulted in structurally, and most likely also functionally different T4aP that may have helped the bacterium to adapt to their specific environment.
The presence of structural variants of PilA (ComP in this study) has previously suggested a functional variation of T4aP in Acinetobacter baumannii [39]. Our data did not allow to reproduce this observation, because the structural variation among PilA proteins is not reflected in differences of their feature architectures (see Fig 1). However, reconciling the phylogenetic distribution of the PilA variants with our results reveals discrepant patterns. For example, while Ab ATCC 19606 T and Ab ACICU differ in their PilA structure [39], they harbor the same T4aP cassette (see Fig 2). This strongly suggests that the evolutionary and functional plasticity of T4aP is substantially higher than anticipated. Thus, an essential part of how pathogenic Acinetobacter isolates interact with their environment, and more specifically with their host, is largely uncharted. It will require highly resolved structural and functional studies to link the various T4aP layouts with lineage-specific differences in T4aP function and to assess the consequences for the bacterial phenotype.

Conclusion
A broad range of bacterial taxa use Type IVa pili for the interaction with their environment, and their functional diversity has earned them the attribute "Swiss Army knife" among bacterial pili [32]. Here, we provided evidence that T4aP are substantially more diverse in Acinetobacter than was hitherto appreciated. Already different isolates within A. baumannii seem to differ in their precise T4aP function, and thus in the way they interact with the human host. This rapid change of pilus function seems to be achieved by an evolutionary concept that resembles an interchangeable tool system where the same handle can convey multiple functions depending on the precise layout of the tool head, here represented by the pilus tip adhesin ComC. Increasing the resolution to trace the functional modification of ComC on the subdomain level reveals the same concept. A conserved structural scaffold formed by the von Willebrand factor A domain appears to be structurally and functionally modified by individual and lineage-specific insertions. On a broader scale, our findings suggest that a substantial extent of functional differences between bacteria isolates that is conveyed by changes on the domain-or sub-domain level rather than by the differential presence/absence of genes remains to be detected. Future comparative genomics approaches that aim to unravel the genetic specifics of pathogens should therefore best extend across different scales of resolution. The goal is to integrate lineage-specific differences on the level of gene clusters, genes, protein feature architectures and 3D structural models into a comprehensive reconstruction of molecular evolution in a functional context.

Data collection
Genome assemblies for 855 isolates from the genus Acinetobacter were retrieved from the RefSeq database release 204 [85] (last accessed March 07, 2021). International clonetype assignments [86] were adopted from [21]. Multilocus sequence typing was performed with MLSTcheck v2.1.17 [87] using the Pasteur scheme [88]. 27 isolates of closely related γ-proteobacteria and two representatives from the genus Neisseria were added as outgroups. The full taxon list is provided as S3 Table.

Phylogenetic analyses
Protein sequences were aligned using MAFFT v7.394 with the linsi method [101]. Phylogenetic gene trees were computed using RAxML v8.2.11 [102] with the rapid bootstrapping algorithm and 100 replicates. The WAG model [103] was selected via the PROTGAMMAAUTO option of RAxML as the best-fitting substitution model. Trees were visualized and annotated using the iTOL website [104]. Alternative topology testing was performed using the AU test [50].

Detection of ancestral recombination
A subset of 33 Acinetobacter isolates together with four outgroup taxa were selected to represent both the phylogenetic diversity and the different ComC layouts in our data. For each taxon, we extracted the genomic region between 5,000 bp upstream and 3,500 bp downstream of comC and aligned the sequences with MAFFT v7.394. The detection of individual genetic lineages together with the prediction of ancestral recombination events was done with fas-tGEAR [51] using default parameters. Shared synteny analyses of the genes annotated in the region across the investigated taxa was assessed using the software Vicinator (https://github. com/ba1/Vicinator) [21] based on the orthology assignments from fDOG.

Culture conditions of bacterial strains and cell lines
All A. baumannii strains used for experiments in this study are listed in Table 2. Strains were grown in Luria-Bertani medium (LB) at 37˚C with 50 μg ml -1 kanamycin or 100 μg ml -1 gentamicin when needed. Human umbilical vein endothelial cells (HUVECs) were extracted from fresh cord veins and cultivated in endothelial growth medium. Medium was supplemented with growth factor mix and 10% fetal calf serum.

Generation of mutants and complementation analysis
The ΔcomC::kanR A. baumannii AYE-T deletion mutant was generated as described by Godeux, Svedholm (111) using primers 1-10 (S4 Table). All mutants were verified by sequencing. Three different constructs were used to complement Ab AYE-T ΔcomC::kanR: (i) full length ComC-The comC gene plus 700 bp of the upstream region were amplified from chromosomal DNA of A. baumannii ATCC 19606 T using primers 11-12 (S4 Table). The amplicon was integrated into pVRL1 [112] using KpnI and XhoI resulting in plasmid pVRL1_comC-19606.

Natural transformation
Wild type and mutant strains were grown in LB medium overnight at 37˚C and diluted to OD 600 0.01 with phosphate buffered saline (PBS). Equal amounts of the bacterial suspension and DNA (100 ng/μl genomic DNA of rifampicin resistant A. baumannii ATCC 19606 T ) were mixed and 2.5 μl of the mixture were applied onto 1 ml of freshly prepared transformation medium (5 g/L tryptone, 2.5 g/L NaCl, 2% [w/v] agarose) in 2 ml reaction tubes. After incubation for 18 hours at 37˚C, cells were resuspended from the medium with PBS. Transformants were selected by plating on selective agar (rifampicin 20 μg ml -1 ).

Twitching motility
Twitching medium (5 g/L tryptone, 2.5 g/L NaCl, 0.5% agarose) was inoculated by stabbing one bacterial colony through the agar to the bottom of the petri dish. Plates were sealed with parafilm to prevent desiccation and incubated at 37˚C for three days. To visualize the cells at the bottom of the petri dish, the agar layer was removed, and cells were stained with 1% [w/v] crystal violet.

Piliation analyses by electron microscopy
To analyze the piliation phenotype, cells were grown on LB agar plates at 37˚C overnight. Cells were prepared for electron microscopy and visualized as previously described [113]. Shadowing of the cells was carried out in an angle of 20˚(unidirectional) and with a thickness of 2 nm platinum/carbon.

Analysis of bacterial adhesion to human endothelial cells
Primary human umbilical cord vein cells (HUVECs) were cultivated in endothelial growth medium supplemented with growth factor mix (ECGM, Promocell) and 10% fetal calf serum (FCS, Sigma-Aldrich) in collagenized 75 cm 2 cell culture flasks using a humidified incubator with a 5% CO2 atmosphere at 37˚C. HUVECs were seeded into six-well plates and infected with A. baumannii (MOI 50) for three hours. The supernatant was removed and cells with adherent bacteria were washed with PBS and detached using a cell scraper. Thereafter, adherent bacteria were quantified by plating serial dilution series. Visualisation and quantification of bacterial adhesion to human endothelial cells was done by fluorescence microscopy as described in [60]. The I->T substitution likely alters the function of the cleavage motif such that it interferes with the processing of FimT. As a consequence, the ability to twitch is lost. Three further non-twitching strains show the canonical prepilin peptidase cleavage motif, however they share an R->H substitution at Pos. 91 in the alignment. Because this is the only difference of these strains to the twitching A. baumannii strains, it is tempting to speculate that also the R->H substitution is sufficient to abolish twitching.